Ion mobility analysis of biological particles

ABSTRACT

A medical diagnostic method and instrumentation system for analyzing noncovalently bonded agglomerated biological particles is described. The method and system comprises: a method of preparation for the biological particles; an electrospray generator; an alpha particle radiation source; a differential mobility analyzer; a particle counter; and data acquisition and analysis means. The medical device is useful for the assessment of human diseases, such as cardiac disease risk and hyperlipidemia, by rapid quantitative analysis of lipoprotein fraction densities. Initially, purification procedures are described to reduce an initial blood sample to an analytical input to the instrument. The measured sizes from the analytical sample are correlated with densities, resulting in a spectrum of lipoprotein densities. The lipoprotein density distribution can then be used to characterize cardiac and other lipid-related health risks.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patentapplication serial No. 60/338,214 filed Nov. 13, 2001, entitled “IonMobility Analysis of Biological Particles”.

STATEMENT REGARDING FEDERAL FUNDING

[0002] This invention was made with U.S. Government support underContract Number DE-AC03-76SF00098 between the U.S. Department of Energyand The Regents of the University of California for the management andoperation of the Lawrence Berkeley National Laboratory. The U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention generally relates to particle size analysisand, further, to analysis of biological particles for diagnosticpurposes utilizing traditional particulate size or mobility measurementdevices. One aspect of the present invention more particularly relatesto medical diagnostics for the quantitative and qualitative analysis oflipoprotein classes and subclasses and their relationship to theassignment of coronary heart disease and other lipid-related healthrisks.

[0005] 2. Description of the Relevant Art

[0006] Introduction

[0007] In clinical practice, lipoprotein particle measurements are usedto assess cardiovascular and other lipid-related health risks, determinetreatment protocols and track the efficacy of treatment regimens.Lipoprotein particles comprise macromolecules that package cholesteroland other biochemicals, enabling them to be transported through theblood stream. The size distribution of lipoprotein particles variesamong individuals due to both genetic and nongenetic influences. Thediameters of lipoprotein particles typically range from about 7 nm toabout 120 nm. In this diameter size range, there exist subfractions ofthe particles that are important predictors of cardiovascular disease.For instance, very low density lipoproteins transport triglycerides inthe blood stream; high very low density lipoprotein levels in the bloodstream are indicative of hypertriglyceremia. These subfractions areidentified by analytical techniques that display the quantity ofmaterial as a function of lipoprotein particle size or density.

[0008] Standard Plasma Lipid and Lipoprotein Cholesterol MeasurementTechniques

[0009] Typical standard lipid measurements include fasting totalcholesterol, triglyceride, as well as HDL and LDL cholesterol.Currently, the most widely used method for measuring LDL cholesterol isthe indirect Friedewald method (Friedewald, et al., Clin. Chem. Vol. 18,pp. 499-502, 1972). The Friedewald assay method requires three steps: 1)determination of plasma triglyceride (TG) and total cholesterol (TC), 2)precipitation of VLDL and LDL, and 3) quantitation of HDL cholesterol(HDLC). Using an estimate for VLDLC as one-fifth of plasma triglycerides$\left( \frac{TG}{5} \right),$

[0010] the LDL cholesterol concentration (LDLC) is calculated by theformula: LDLC=TC−(HDLC+VLDLC). While generally useful, the Friedewaldmethod is limited in its accuracy in specific cases. Errors can occur inany of the three steps, in part because this method requires thatdifferent procedures be used in each step. The Friedewald method is to adegree indirect, as it presumes that VLDLC concentration is one-fifththat of plasma triglycerides. When the VLDL of some patients deviatesfrom this ratio, further inaccuracies occur. Ultracentrifugation must beemployed for separation and subsequent determination of LDL cholesterolfor some samples, since the Friedewald method cannot be used forpatients with TG over 400 mg/dL.

[0011] Procedures for Lipoprotein Subspecies Analysis

[0012] Presently, the predominant methods for lipoprotein subspeciesanalysis include nuclear magnetic resonance, the vertical auto profile,and Electrophoretic gel separation. Each of these methods will bebriefly discussed below.

[0013] Nuclear Magnetic Resonance

[0014] Otvos teaches a nuclear magnetic resonance (NMR) procedure fordetermining the concentrations of lipoprotein subclasses, which hasgreater accuracy than Friedewald (U.S. Pat. No. 5,343,389, issued Aug.30, 1994). Otvos initially obtains the NMR chemical shift spectrum of ablood plasma or serum sample. The observed spectrum of the entire plasmasample is then matched with the known weighted sums of NMR spectra oflipoprotein subclasses, which are stored in a computer software program.The weight factors that give the best fit between the sample spectrumand the calculated spectrum are then used to estimate the concentrationsof constituent lipoprotein subclasses in the blood sample. Thisprocedure has the additional advantage of being rapid.

[0015] Vertical Auto Profile

[0016] Another lipoprotein subfraction determination method that is usedclinically is the Vertical Auto Profile (VAP), (Kulkarni, et al., J.Lip. Res., Vol. 35, pp. 159-168, 1994). In the Vertical Auto Profilemethod, a flow analyzer is used for the enzymatic analysis ofcholesterol in lipoprotein classes separated by a short spin singlevertical ultracentrifugation, with subsequent spectrophotometry andsoftware analysis of the resulting data. While a useful advance, thistechnique does not resolve the LDL into all seven subspecies identifiedby electrophoretic gradient gel separation.

[0017] Electrophoretic Gradient Gel Separation

[0018] The gel separation method is demonstrated in U.S. Pat. No.5,925,229, issued Jul. 20, 1999, by R. M. Krauss, et al., entitled “LowDensity Lipoprotein Fraction Assay for Cardiac Disease Risk,” (the '229patent), which is hereby incorporated by reference. In the '229 patent,a gradient gel electrophoresis procedure for the separation of LDLsubclasses is disclosed. The LDL fractions are separated by gradient gelelectrophoresis, producing results that are comparable to those obtainedby ultracentrifuge. This method generates a fine resolution of LDLsubclasses, and is used principally by research laboratories. However,gradient gel electrophoresis can take many hours to complete. It wouldbe useful if gradient gel electrophoresis separation times could beshortened and the analysis simplified so that high resolution lipidanalysis could be used in clinical laboratories as part of a routinescreening of blood samples, and to assign a risk factor for coronaryartery disease.

[0019] The gel separation method, which depends on uniform staining ofall components that are subsequently optically measured, suffers fromnonuniform chromogenicity. That is, not all lipoprotein particles stainequally well. The differential stain uptake produces erroneousquantitative results, in that a less staining peak may be read at alower value than is actually present. Additionally, the nonuniformchromogenicity can result in erroneous qualitative results, in thatmeasured peaks may be skewed to a sufficient degree as to confuse oneclass or subclass of lipoprotein with another.

[0020] A high-resolution assay for measuring all subclasses of LDL aswell as VLDL, IDL, HDL, and chylomicron particles that would beaccurate, direct, and complete, would be an important innovation inlipid measurement technology. If inexpensive and convenient, such anassay could be employed not only in research laboratories, but also in aclinical laboratory setting. Ideally, clinicians could use thisinformation to improve current estimation of coronary disease risk andmake appropriate medical risk management decisions based on the assay.

SUMMARY OF THE INVENTION

[0021] This invention provides: 1) a method for preparing plasma samplesfor differential mobility analysis (“DMA”), 2) a technique for measuringthe size or density distribution of biological particles (preferablylipoprotein particles) based on the measured particle mobility countingrate for the number of biological particles counted per second at aspecific selected mobility as a function of size,$\left. \frac{n^{+}}{t} \right|_{S},$

[0022] or density, $\left. \frac{n^{+}}{t} \right|_{\rho},$

[0023] of ionized biological particles sprayed into a fluid (such asair), 3) a DMA device for measuring the number of biological particlesin each size class and subclass, 4) an algorithm for predicting the riskof coronary heart disease (CHD) using the biological particle(preferably lipoprotein) size or density distribution informationcompared to reference data for low risk, intermediate risk, and highrisk patients, and 5) a particular method for determining whether asample corresponds to a Type A, AB, or B pattern, which respectively hasan associated low risk, an intermediate risk, and a high risk for CHD.Blood plasma samples are processed to separate all classes andsubclasses of lipoprotein particles from bulk blood serum using stepsdesigned ultimately for transferring the particles into the gas phasefor subsequent mobility analysis. The technique preserves lipoproteinparticle composition and size, and introduces no contamination into thelipoprotein particles. The electrospray process initially produceselectrospray droplets with many different charge states. Thesemultiply-charged droplets are passed in close proximity to an alpharadiation source, which produces alpha particles, or free secondaryelectrons, that reduces the charge state of the electrospray droplets toeither neutral or single positive charges. The charged droplets mayalternatively be treated in other ways to achieve a uniform charge stateof no more than a single positive charge.

[0024] Subsequent volatilization of the droplet diluent results inindividual lipoprotein particles having either a neutral or a singlepositive charge. By scanning a high voltage selection voltage thatmodifies a mobility selection, lipoprotein particle gas-phase mobilitycan be measured over a range of mobilities. When the scanned mobilitiesare counted over a scan range, a mobility distribution can be developed.Personnel skilled in reading the lipoprotein mobility distribution canmake direct conclusions regarding the cardiac risk for the patient.

[0025] An inverse linear relationship between lipoprotein mobility andsize allows the mobility distribution data can be converted to aparticle size distribution, thereby providing a way to display aspectrum, or distribution, of the number of particles counted per unittime vs. particle size. Given a constant time basis, this measurementcan be regarded as number of particles vs. particle size, and can berelated back to a quantitative fractional distribution of lipoproteinsin the original sample. The resulting lipoprotein particle sizedistribution is comprised of component regions. These regions, in turn,distinguish the quantity and types of lipoprotein particles in theoriginal serum sample. By determining the relative location of certainpeaks in certain of the regions, a prediction can be made as to thecardiac risk. The size distribution may additionally be converted into adistribution of the number of particles vs. density for more traditionalcomparison of lipoprotein densities.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 is an overview of the ion mobility analysis of biologicalparticles hardware and analytical sequence.

[0027]FIG. 2 is a table of major lipoprotein classes, subclasses,densities and particle sizes as reported from gel electrophoresisresults.

[0028]FIG. 3 is a close up cross-sectional view of a properly operatingaxisymmetric electrospray capillary with a stable Taylor cone.

[0029]FIG. 4 is a cross sectional view through the centerline of anaxisymmetric differential mobility analyzer.

[0030]FIG. 5 is a scanning differential mobility analyzer output ofmass-offset lipoprotein size distributions obtained from sixindividuals.

[0031]FIG. 6 is a graph indicating the approximately inverse linearrelationship between lipoprotein particle diameter, and the estimatedlipoprotein sample density.

[0032]FIG. 7 is a graph indicating the approximately linear relationshipbetween lipoprotein particle count, and the corresponding lipoproteinsample particle count.

[0033]FIG. 8 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.000 g/cm³,corresponding to VLDL I.

[0034]FIG. 9 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.003 g/cm³,corresponding to VLDL I.

[0035]FIG. 10 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.005 g/cm³,corresponding to VLDL I.

[0036]FIG. 11 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0075 g/cm³,corresponding to VLDL II.

[0037]FIG. 12 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0105 g/cm³,corresponding to IDL I.

[0038]FIG. 13 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0161 g/cm³,corresponding to IDL II.

[0039]FIG. 14 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0217 g/cm³,corresponding to LDL I.

[0040]FIG. 15 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0273 g/cm³,corresponding to LDL IIa.

[0041]FIG. 16 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0330 g/cm³;corresponding to LDL IIb.

[0042]FIG. 17 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0386 g/cm³,corresponding to LDL IIIa.

[0043]FIG. 18 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0442 g/cm³,corresponding to LDL IVa.

[0044]FIG. 19 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0498 g/cm³,corresponding to LDL IVa.

[0045]FIG. 20 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0692 g/cm³,corresponding to HDL IIb.

[0046]FIG. 21 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0767 g/cm³,corresponding to HDL IIb.

[0047]FIG. 22 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.0844 g/cm³,corresponding to HDL IIb.

[0048]FIG. 23 is a differential mobility spectral scan of a sample oflipoprotein having an average density of about 1.090 g/cm³,corresponding to HDL IIb.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0049] A. Definitions

[0050] “Biological particle” means a material having a non-covalentlybound assembly of molecules derived from a living source. Examples arelipoprotein particles assembled from apolipoproteins and lipids; viralcomponents assembled from non-covalently bound coat proteins andglycoproteins; immune complexes assembled from antibodies and theircognate antigens, etc., but not entire cells.

[0051] “Physiological sample” means a sample obtained from an organism,such as blood, tissue, pulp, cytoplasm, etc.

[0052] “CHD” means coronary heart disease.

[0053] “VLDL, IDL, LDL, and HDL” are described by class name, acronym,subclass, and density range as shown in FIG. 2.

[0054] “Chylomicrons” means biological particles of size 70-120 nm, withcorresponding densities of less than 1.006 g/mL.

[0055] “Differential Mobility Analyzer” means a device for classifyingcharged particles on the basis of their ion electrical mobility. Whenthe particles have a known uniform charge, the size of the particlesclassified may be determined from their mobility.

[0056] “Lp(a)” means biological particles consisting of LDL covalentlyattached to the protein lipoprotein A.

[0057] “Lipoprotein particles” means particles obtained from mammalianblood, comprising apolipoproteins biologically assembled withnoncovalent bonds to package cholesterol and lipids. Lipoproteinparticles preferably refer to biological particles having a size rangeof 7 to 1200 nm. Lipoprotein particles, as used herein, essentiallyinclude VLDL, IDL, LDL, HDL and chylomicrons.

[0058] “Centrifugation” means separation or analysis of substances in asolution as a function of density and density-related molecular weightby subjecting the solution to a centrifugal force generated byhigh-speed rotation in an appropriate instrument.

[0059] “Pattern A” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating a relativelylow risk for CHD, as illustrated in FIG. 5.

[0060] “Pattern B” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating a relativelyhigh risk for CHD, as illustrated in FIG. 5.

[0061] “Pattern AB” is the designation applied to individuals having acharacteristic lipoprotein particle distribution indicating anintermediate risk for CHD, as illustrated in FIG. 5. Pattern AB has adistribution with some of the characteristics of both Pattern A andPattern B.

[0062] “Predisposition” as used herein is substantially synonymous withrisk, inclination, tendency, predilection, or susceptibility.

[0063] “Reference Size Distribution” means a lipoprotein particle sizedistribution such as Patterns A, B, or AB as further discussed inconnection with FIG. 5.

[0064] “Computer readable medium” means any source of organizedinformation that may be processed by a computer, including but notlimited to: a magnetically readable storage system; optically readablestorage media such as punch cards or printed matter readable by directmethods, optical reflectance and transmission scanning, or methods ofoptical character recognition; other optical storage media such as acompact disc (CD), digital versatile disc (DVD), rewritable CD and/orDVD; electrically readable media such as programmable read only memories(PROMs), electrically erasable programmable read only memories(EEPROMs), field programmable gate arrays (FPGAs), flash random accessmemory (flash RAM); and remotely transmitted information transmitted byelectromagnetic or optical methods.

[0065] “Distribution” means a generalized function of one or morevariables, and commonly depicted as a scatter chart, graph, plot, orhistogram.

[0066] B. Overview of Ion Mobility Analysis of Biological Particles

[0067]FIG. 1 depicts the major functional components involved inbiological particle ion mobility analysis. A sample is placed in amicrocentrifuge tube 410, which is then placed in an centrifuge 420 toremove undesirable components, such as Lp(a), and other cellularcomponents as described below in Section D “Preparation of PurifiedLipoprotein Particle Samples.” After removal of cellular components andLp(a), as well as further density ultracentrifugation to select onlylipoprotein materials, a sample solution 455 is placed in a testingmicrocentrifuge tube 450, which is in turn placed in a pressurizedchamber 460. A high voltage variable power supply 430, positively biasesthe pressurized chamber 460, and the sample solution 455, through a Ptwire 435. The positive bias and higher relative pressure causes acapillary 100 to form a Taylor cone 150 emitting particle droplets fromthe pressurized chamber 460. FIG. 3 more fully details the capillary100.

[0068] Still referring to FIG. 1, once the droplets are formed, a drygas 210 propels the droplets into an emission region of an alpharadiation source 480, which reduces the charge state of the droplets tono more than one positive charge per droplet as the droplets containingcomponents other than the volatile diluent dry to single particles. Thecharged droplets may alternatively be treated with other methods toachieve a uniform charge state of no more than a single positive charge.One such other method is to use an alternating current corona, whichproduces secondary electrons having the same charge state reduction asan alpha source.

[0069] After charge reduction, the dry gas 210 propels the particlesinto a differential mobility analyzer 200, which is shown in more detailin FIG. 4. A laminar flow excess gas 220, as well as an additional drygas 230 flow is introduced to match the velocity of the dry gas 210flow. By varying a high voltage power supply 205, the particles carriedby the combined flows are selected into a mobility selected particle 240flow, which in turn flows into a particle counter 490.

[0070] Particle counter 490 is preferably in communication with acomputer system (not shown) having storage capability onto one or morecomputer readable media for recording a scanned distribution output ofthe differently sized particles. As discussed below under Section F“Differential Mobility Analysis,” the single positively chargedparticles, the fixed dry gas 210 and additional dry gas 230 flows, andparticular voltage setting of the scanned high voltage power supply 205,classifies a selected particle stream 285 to enter into an exitingannular selection slit 290, the selected particle stream 285 havingparticles with a particular mobility and corresponding particle size.

[0071] One feature of this invention is that loose, non-covalentlybonded biological particles can be processed through this system withoutlosing their biological identity or breaking apart. In particular,lipoprotein particles (e.g. VLDL, IDL, LDL, and HDL and theirsubclasses) can be processed in such a fashion so that their particlemobilities, and hence size and density, can be quickly determined,without lipoprotein particle breakdown.

[0072] Once a relationship is known between particle size and density,size or mobility distributions may be converted into distributions of: aparticle mass, a density μg/cm³ of original plasma, a number ofparticles in a size interval, and an amount of particle mass in a sizeinterval.

[0073] C. Lipoprotein Particles

[0074] Lipoproteins are complex macromolecules found in human bloodplasma that package lipids into lipoprotein particles. Among otherfunctions, lipoprotein particles in the circulatory system transportcholesterol for cellular use. Lipoprotein particles are subdivided intoa variety of classes and subclasses based on density as well as particlesize. Lipoprotein particle density can be determined directly byequilibrium density ultracentrifugation and analyticultracentrifugation. Lipoprotein particle density may also be determinedindirectly based on particle size and a known relationship betweenparticle size and density. Particle size may be determined by severalmethods including gel electrophoresis.

[0075]FIG. 2 is a table of the present standard classes and subclassesthat have been assigned to various lipoprotein fractions usingtraditional gel electrophoresis measurements: very low densitylipoproteins (VLDLs) with subclasses VLDL I and II, intermediate densitylipoproteins (IDLs) with subclasses IDL I and II, low densitylipoproteins (LDLs) and high density lipoproteins (HDLs), whichtypically includes several subclasses, such as HDL IIa, IIb, IIIa, IIIb,and IIIc.

[0076] Chylomicrons are not included in FIG. 2, as chylomicrons have notbeen found to have any clinical significance in the prediction of heartdisease.

[0077] Ultracentrifugally isolated lipoprotein particles can be analyzedfor flotation properties by analytic ultracentrifugation in twodifferent salt density backgrounds, allowing for the determination ofhydrated LDL particle density, as shown in Lindgren, et. al, BloodLipids and Lipoproteins: Quantitation Composition and Metabolism, Ed. G.L. Nelson, Wiley, 1992, p. 181-274, which is incorporated herein byreference.

[0078] The LDL class, as indicated in FIG. 2, can be further dividedinto seven subclasses based on density or particle diameter by using apreparative separation technique known as equilibrium density gradientultracentrifugation (EDGU). It is known that elevated levels of specificLDL subclasses, LDL-IIIa, IIIb, IVa and IVb, is a clinical finding whichcorrelates closely with increased risk for CHD, includingatherosclerosis.

[0079] Determination of the total serum cholesterol level and the levelsof cholesterol in the LDL and HDL fractions are routinely used asdiagnostic tests for coronary heart disease risk. Lipoprotein class andsubclass distribution is a more predictive test, however, since it isexpensive and time-consuming, it is typically ordered by physicians onlyfor a limited number of patients.

[0080] It should be noted that the values used in FIG. 2 for sizes aredetermined by gel electrophoresis methods. With the ion mobility methodsdisclosed here, it has been observed that all measurements oflipoprotein particle diameter obtained with ion mobility are shifted tosmaller diameters compared to the data obtained with gelelectrophoresis. It is surmised that this difference is caused bycalibration of the gels. The shift appears to be linearly related withapproximately:

0.86*gel diameter=ion mobility diameter

[0081] The observed differences between ion mobility diameters and gelelectrophoresis diameters may also be due to the fact that thelipoprotein particles are distorted as they bump into the gel matrixunder the influence of the electrophoresis gel's intrinsic impressedelectric field. The size difference may also be due to historical dataused to convert particle density (obtained from analytic ultracentrifugeseparations) to particle size obtained from electron microscopy. FIG. 2presents the lipoprotein sizes accepted for gel electrophoresis runs aspublished in common scientific literature. It is the inventors' opinionthat ion mobility sizes may be more accurate than gel sizes, however,this needs to be confirmed.

[0082] D. Preparation of Purified Lipoprotein Particle Samples

[0083] Generally, sample preparation comprises the following steps:taking a blood sample, separation of the blood plasma from the cellularmatter, removal of non-lipoprotein proteins from the plasma by one ofseveral methods, such as ultracentrifugation or adsorption on affinitygels, dialysis filtration to remove density adjusting components thatwould affect particle volatility and/or size, addition of volatilereagents to facilitate electrospray droplet formation, dilution oflipoprotein particle sample solution, and adjustment of sample solutionpH.

[0084] A 50 to 100 μL fasting blood sample is initially taken.Chylomicrons are not typically present in people who have been fastingfor a period of at least 12 hours, so overlap of VLDL particle sizes andchylomicron sizes is eliminated by fasting. The sample is then initiallyspun in a centrifuge. The sample is preferably spun for approximately 10minutes at 2000 gravities, sufficient to remove the cellular componentsfrom the sample. During this process, the more dense cellular componentsstratify at the bottom of the sample. A remaining less dense plasmaspecimen on top is then drawn off.

[0085] The plasma specimen is then density-adjusted to a specificdensity using high purity solutions of sodium chloride (NaCl) and sodiumbromide (NaBr). In one embodiment, the specific density is chosen to begreater than or equal to the highest density of the lipoprotein materialto be analyzed, so that the lipoprotein material floats when densitystratified. These densities may be found in the FIG. 2 table oflipoprotein classes, subclasses, densities, and sizes. By sequentiallycentrifuging from lowest density to highest density of the densityadjustment, the various classes and subclasses of lipoproteins may besequentially extracted.

[0086] In the preferred method used in this invention, instead of aseries of sequential density adjustments followed by centrifugation,just a single density adjustment using NaCl and NaBr salts is performed.A single specific density is chosen for density adjustment in thepreferred embodiment. The sample density adjustment can be selectedwithin the range of 1-1.21 g/mL according to the densities in FIG. 2 toseparate a class of lipoproteins having equal or lesser density. In thepreferred embodiment, a density adjustment to 1.21 g/mL is made. In thismanner, all HDLs, IDLs, LDLs, and VLDLs are simultaneously extracted,since they have densities less than 1.21 g/mL.

[0087] The density-adjusted plasma sample is then ultracentrifuged in acommon commercially available 100 μL ultracentrifuge tube forapproximately 18 hours at 100,000 gravities, or 10⁶ m/s² to separate thenon-lipoprotein proteins from the lipoprotein particles. Non-lipoproteinproteins, particularly albumin, are then removed from the plasmaspecimen, preferably by this ultracentrifugation step. The lipoproteinparticles float to the top of the sample during ultracentrifugation.

[0088] Lp(a) is another type of lipoprotein particle found in serumhaving a molecular composition distinct from IDL and LDL particles.Lp(a) has a particle size that overlaps with LDL and IDL particles andtherefore will interfere with particle size analysis when Lp(a)particles are present in serum. Although some patients have naturallyoccurring low Lp(a) concentrations, it is a good practice to remove theLp(a) prior to LDL particle size measurements to preclude otherwiseinaccurate measurements for those patients who do have significant Lp(a)concentrations. In this manner, any potential Lp(a) size interferenceproblem is avoided.

[0089] In sample solutions where Lp(a) has not been removed, up to halfof the LDL measurement can be comprised of Lp(a). Therefore, beforeelectrospray analysis Lp(a) is preferably removed from the plasmasample. This removal can be achieved by solid phase absorption of theLp(a) protein using either lectin or specific antibodies attached to amatrix. Lp(a) concentration can then be measured separately (e.g. byimmunoassay) or by the difference between the immunoassay results usingabsorbed and unabsorbed plasma. Lp(a) is most preferably removed bylectin affinity chromatography. The Lp(a) removal step preferablyfollows density adjustment and ultracentrifugation. It precedes dialysisof the selected 1.21 g/mL fraction.

[0090] The lowest density, top portion, of the ultracentrifuged sampleis then drawn off for subsequent dialysis as a liquid sample containinglipoprotein particles. This liquid is then placed behind a 10,000 Daltonmolecular weight cutoff filter, to retain the lipoprotein particlesbehind the filter, yet freely allow diffusion of H₂O, NaCl and NaBrionic components. The filter, with the liquid containing the densityadjusting NaCl and NaBr, is immersed in 0.22 μm particle-filtered, 18Meg-ohm deionized water to allow the NaCl and NaBr to reachconcentration equilibrium with the surrounding water. In this fashion,the NaCl and NaBr, which otherwise would have affected particle weightduring mobility measurement, are removed, or dialyzed. In thelaboratory, it has been found sufficient to place the cutoff membrane incontact with water, and simply deposit a drop of sample to the top ofthe membrane. The NaCl and NaBr levels in the drop of sample then reachequilibrium with the water diluent.

[0091] The sample solution is then prepared in a volatile aqueous bufferat sufficiently near neutral pH (pH 7.0), so as to prevent thelipoprotein particles from disintegrating into one or more of theirconstituent molecules. The preferred buffer solution is 20-30 mM,preferably 25 mM, ammonium acetate in particle-filtered 18 Meg-ohmdeionized water with pH adjusted to neutral with high purity ammoniumhydroxide. It is critical to prepare the ammonium acetate and ammoniumhydroxide solutions with very high purity reagents in order to preventcontamination of the lipoprotein particles, which would subsequentlyaffect the particle mobility measurements.

[0092] The volatile aqueous buffer serves three principal functions: 1)to dilute the sample sufficiently so that, when electrosprayed, only onelipoprotein particle is contained within each electrospray droplet, 2)to provide for electrical conductivity so that the capillary bore samplecontents are uniformly at approximate equipotential with the highvoltage power source, and 3) to impart a higher vapor pressure to thebulk solution, thereby improving volatility of the diluted sample andhastening droplet drying time. Although ammonium acetate has been usedhere, people skilled in the art could arrive at several alternativeformulations achieving the same three functions.

[0093] The amount of insoluble material and the residue remaining afterdilution and subsequent volatilization are important specifications forchoosing a particular quality of reagent. These quantities should beminimized; preferably each reagent should have less than 0.005 weightpercent of impurities. Subsequently, the lipoprotein sample is eitherprepared in solution with the reagent or diluted with this reagent toabout 10¹¹ particles per mL or less.

[0094] At lipoprotein particle concentrations of less than 10¹¹particles per mL, no more than one lipoprotein particle should bepresent in a single electrospray droplet. By designing the dilutionprocess to have only one lipoprotein particle in an electrospraydroplet, a potential aliasing problem of artificially combininglipoprotein particle clusters, and thereby detecting the lumped clusteras a larger lipoprotein, is avoided. Restating this issue, if two smallweight lipoprotein particles arrive in a single droplet, then theresulting size measurement of the compound particle is much larger, andis not representative of the original lipoprotein sample. Thus, ifaliasing were to occur, two or more small HDL particles could bemeasured as a single VLDL particle, distorting the mobility measurement,and further indicating an incorrect corresponding particle sizedistribution.

[0095] The lipoprotein particles suspended in the ammonium acetatesolution are then electrosprayed. The electrospray is fed by pumping thesample solution at a rate of about 50 nL per minute through a smallcapillary tube, preferably an Osage number 062442 capillary. Between 20and 50 μL of sample solution is placed in a microcentrifuge tube and theentrance to a silica capillary having a 20 μm interior diameter isplaced into this sample solution. The microcentrifuge sample tube andthe entrance end to the capillary are sealed in a positive pressurecontainer. A positive differential pressure, on the order of 3 psig, isapplied across the capillary, producing a flow of sample solutionthrough the capillary. The outlet side of the capillary, maintained atapproximately ambient atmospheric pressure, is inserted into anelectrospray droplet generator, preferably a TSI Model 3480 ElectrosprayAerosol Generator (TSI, Incorporated, St. Paul, Minn.).

[0096] E. Electrospray of Samples

[0097] The electrospray capillary used in this invention is shownaxisymmetrically in FIG. 3. The capillary body 100 has a hollow core,through which a sample solution 130 is transported. It is preferred touse a capillary 100 that has been passivated with a coating such as amethyl derivatization along the hollow core of the interior bore 110 ofthe capillary 100, so that substances found in the sample solution 130are not adsorbed as the sample solution 130 is electrosprayed, as suchadhesion would tend to clog the capillary, rendering it unusable untilsuccessfully cleaned. It has been found that a capillary derivatizedwith a methylating reagent replaces silanol and Si—O— groups on theinterior bore 110 with methyl groups, which thereby minimizes adsorptionof sample onto the capillary, and subsequent clogging.

[0098] Stable electrosprays are obtained when the capillary 100 is about25 cm long and a positive voltage of about 2 kV is applied to the sample130. This is accomplished, as shown in FIG. 1, by placing a Pt wire 435into the testing microcentrifuge tube 450 and in electrical contact withthe sample solution 435. The Pt wire 435 is connected to the positivepolarity of a high voltage variable power supply 430. It is alsonecessary to taper the wall of the outlet end of the capillary 120 toproduce a truncated flat-tipped cone 140 with about a 90° includedtaper. The tapered area of the capillary thus resembles a truncatedcone.

[0099] Refer now to FIG. 3. The tapered outlet end 120 of the capillary100 is produced by placing a capillary 100 securely into a pin-visemounted on a 50 rpm DC motor, and subsequently grinding an approximately45° angle from the longitudinal direction of the capillary.

[0100] Electrospray occurs at the flat-topped tapered outlet end of thecapillary 120. The meniscus of the exiting sample 130 liquid takes onthe characteristic shape of a Taylor cone 150, typical of stableelectrosprays, but only a sharp-tipped cone 160 can be seen during theelectrospray of highly conductive liquids typically used because thedroplet stream 170 that forms is comprised of droplets too tiny toscatter light and thus be microscopically observed.

[0101] The droplet stream 170 is carried into a small chamber by alaminar flow of CO₂ and air, established according to TSI factoryrecommendations, where they are exposed to an alpha radiation source 480(as previously shown in FIG. 1), which, as the droplet diluentevaporates, lowers the droplet net charge state to zero or one. Theinitial droplet size is typically approximately 150 nm in diameter.

[0102] As discussed in Section B “Overview of Ion Mobility Analysis ofBiological Particles,” the droplets dry rapidly in the flow of CO₂ andair and desolvate forming neutral or singly charged lipoproteinparticles. The lipoprotein particles carry the same amount of charge asdid the droplets that were initially electrosprayed. In typicalimplementations, drying and charge reduction may take place concurrentlyto prevent Coulomb forces breaking apart droplets, and here lipoproteinparticles. The resulting gas-borne particles are primarily chargeneutral or carry one positive charge. The alpha-source charge reductionprocess produces a reliable, well-characterized stream of chargedparticles. In this case, well characterized means that, although thefraction of singly charged particles depends on particle diameter, therelationship between diameter and the fraction of the particles carryinga single charge is well established. The electrospray aerosol generatordelivers neutral and singly-charge lipoprotein particles to the input ofa differential mobility analyzer 200 where the size distribution of theparticles is determined.

[0103] Other methods may be used to ensure that an entering stream ofcharged particles exits with particles having no more than a singlepositive charge. One of these methods include using an alternatingcurrent corona to produce secondary electrons having the same chargestate reduction as an alpha radiation source.

[0104] F. Differential Mobility Analysis

[0105] Ion electrical mobility analysis is a technique to determine thesize of a charged particle undergoing analysis when the charged particleis exposed to an electric field. Below follows the analytical methodused to determine the size of the charged particle.

[0106] Ion electrical mobility is a physical property of an ion and isrelated to the velocity an ion acquires when it is subjected to anelectrical field. Electrical mobility, Z, is defined as$Z = \frac{V}{E}$

[0107] where V=terminal velocity and E=electrical field causing particlemotion. Furthermore, particle diameter can be obtained from$Z = \frac{{neC}_{c}}{3\quad \pi \quad \eta \quad d}$

[0108] where n=number of charges on the particle (in this case a singlecharge), e=1.6×10⁻¹⁹ coulombs/charge, C_(c)=particle size dependent slipcorrection factor, η=gas viscosity, and d=particle diameter. Solving ford, we obtain$d = {\frac{{neC}_{c}}{3\quad \pi \quad \eta}\quad \frac{E}{V}}$

[0109] Thus we obtain an explicit relationship for particle diameter asa function of known parameters. By setting the parameters to differentvalues, different particle diameters of the charged particles may beselected as further described below. In particular, it is easiest tovary E, the electric field strength acting upon the charged particle.

[0110] A differential mobility analyzer separates the charged inputlipoprotein particles according the their diameter and charge. Apreferred differential mobility analyzer is a TSI model 3080Electrostatic Classifier.

[0111] Now referring to FIG. 4, a cross section through the centerlineof a typical axisymmetric differential mobility analyzer 200 is shown.The analyzer is designed and operated to transmit only singly chargedpositive ions of a particular size 285 through an exiting annularselection slit 290 in the instrument. For purposes of illustration thereare several particles shown that are not at the correct selection size285: neutral particles 295, and charged smaller particles 260 andcharged larger particles 280.

[0112] The differential mobility analyzer 200 is housed in a cylindercanister 275 having a hollow cylindrical area 245 with a centrallyplaced tube 270. The centrally placed tube 270 comprises an annularselection slit 290 through which particles (e.g. 285 from the hollowcylindrical area 245) pass when a certain electric field exists.Particles 285 enter the annular selection slit 290 as a function of thesize of the particle and the electromotive force applied to theparticle. An electromotive force results from the application of aselection voltage applied from high voltage source 205 to centrallyplaced tube 270 and cylinder canister 275. Once a particle 285 enterscentrally placed tube 270 through selection slit 290, the gas flow 230through the centrally placed tube 270 carries particle-laden gas flow240 to a particle-counting device 490 (as shown in FIG. 1).

[0113] In FIG. 4, four different types of particles are shown forpurposes of illustration. The four different symbols represent singlycharged lipoprotein particles having three different sizes 260, 280, and285, and an uncharged particle 295. When a high voltage source 205 isapplied between the centrally placed tube 270 and the outer cylinder275, the positively charged particles begin to move towards thecentrally placed tube 270 at a radial velocity determined by the balancebetween the forces exerted by the electric field strength and theviscous resisting force due to the drag of the particle moving throughthe ambient medium.

[0114] Since the viscous drag is related to the size of the particle,for the same electromotive force, a smaller particle, having a smallercross sectional area, will have a smaller drag, will be most affected bythe electromotive force, and will consequently have a higher mobility.Correspondingly, a larger particle will have a larger cross sectionalarea, and will be least affected by the same electromotive force, andconsequently have a lower mobility.

[0115] The axial gas velocity moving from the top of the analyzer 225 tothe bottom of the analyzer 235 influences the location where theparticles impinge the centrally placed tube 270 by vector velocitycomponent addition. FIG. 4 shows that one of the types of particle ions285 has a mobility that deposits it into a detection slit 290 that leadsthe selected ions away to a detector 490. That is, only a smalldistribution of particle sizes distributed about the selected particlesize exits detection slit 290 at a specified high voltage 205 at aspecific laminar gas velocity in the hollow cylindrical area 245.

[0116] Particle laden dry gas 210 introduces the particles into thedifferential mobility analyzer 200, with a laminar excess gas flow 220introduced so as to minimize turbulent eddies of any sort. An additionaldry gas 230 is introduced at the top of the analyzer 225, ultimatelyexiting the bottom of the analyzer 235 as mobility-classified particleflow 240.

[0117] In FIG. 4, only those particles with a particular size 285 arepresently being selected. Smaller sized particles 260 impinge on thecenter tube before the detection slit 290. Larger sized particles 280either impinge after the slot, or are carried away with the bulk gasflow, but in neither case are measured. Uncharged particles 295, areunaffected by the electromotive force, thus pass through the exit gasstream 250 without passing through the detection slit 290. Mobilityspectra may then be obtained by scanning the high voltage source 205applied to the centrally placed tube 270 and cylinder canister 275through a range of voltages corresponding to the sizes of the particlesof interest. As the differential voltage is scanned each of the threeions will be guided to hit the slit and pass on to a detector locateddownstream. In this example, the small 260, medium 285, and largeparticles 280 may respectively represent HDL, LDL, and VLDL.

[0118] The selectable differential mobility analyzer operates bycounting the number of charged biological particle ions, dn⁺, in adefined sampling time, dt, at a specific selected size, s, resulting ina size count rate output of$\left. \frac{n^{+}}{t} \middle| {}_{S}. \right.$

[0119] The selectable differential mobility analyzer can be scanned overthe desired specific size range to result in an output of chargedbiological particle ions counted in the defined sampling time versussize. Since the relationship between particle size and density is wellunderstood, the size count rate output above can in turn be converted todata set of the number of biological particles counted per second at aspecific density, $\left. \frac{n^{+}}{t} \right|_{\rho},$

[0120] versus density, ρ.

[0121] We now look at the particular case where the biological particlesare lipoproteins. In this case, a scanning size range of 3 nm to 120 nmis appropriate, corresponding to the size of the least and most denselipoproteins present in human patients. Once the scanned data set isproduced in the traditional format of the number of lipoproteinparticles counted per second at a particular size, the specific quantityof lipoprotein versus density can be calculated. By numericallyintegrating the amount of lipoprotein within a density rangecorresponding to the various lipoprotein classes and subclasses,traditional analysis of cardiac heart risk may be assessed.

[0122] G. Data Acquisition and Analysis

[0123] The detector 490 used to record the mobility spectrum is acondensation particle detector, preferably a Model 3025A CondensationParticle Detector from TSI. The condensation particle detector drawsparticles such as lipoprotein particles through a supersaturated vaporcondensation chamber and allows the vapor to condense onto theparticles, which act as condensation nucleation sites. The device issensitive at the single particle level because every lipoproteinparticle nucleates condensation and gives birth to a solvent droplet. Asingle lipoprotein particle becomes completely covered inside eachcondensation droplet. The condensation process, in effect, magnifies thesize of lipoprotein particles and causes them to grow from 10's of nm indiameter to several μm in diameter.

[0124] The μm-scale droplets efficiently scatter light into aphotodetector as they pass through a laser beam. The resultant dropletsscatter light sufficiently to allow for counting individual droplets bymeasuring changes in transmission or reflectance (scatter) in the laserbeam or other light source. Since only a single lipoprotein particle iscontained in each droplet, this then becomes a method for indirectlycounting single lipoprotein particles.

[0125] Lipoprotein particle density measurement in the near-atmosphericgas-phase by particle mobility measurement provides a new way to rapidlydetermine the size distribution of the lipoprotein particles. Referringto FIG. 2, a table is shown including the density and particle size ofthe major lipoprotein subfractions. Since the size and density oflipoprotein particles correlate very well, it is straightforward tocalculate one in terms of the other. This correlation may be shown in aplot of measured diameters and corresponding densities, which are curvefit by a line. This is shown in FIG. 6 and discussed in Section J“Linearity Versus Density.” Thus any lipoprotein particle mobilitymeasurement of a particular size can be related to its correspondingparticle density.

[0126] A mobility spectrum of six typical lipoprotein samples obtainedfrom human blood is presented in FIG. 5. Each lipoprotein particlesample solution, prepared as previously discussed, was diluted to lessthan 10¹¹ lipoprotein particles per mL with a solution of 25 milliMolarammonium acetate. The sample was electrosprayed at a rate of 50 nL permin using a positive 1850 volts to create a well-formed, stableelectrospray. A stable electrospray refers to a lack of visiblefluctuations in the Taylor cone. A flow of 0.5 L per min CO₂ wascombined with 1.0 L per min of dry air to carry the electrospraydroplets through a charge neutralizer, through a differential mobilityanalyzer, and into a condensation particle detector. The resultingspectrum of lipoprotein particles is displayed in FIG. 5, which will bemore thoroughly described below.

[0127] Recall that there is a scalar difference between the gelelectrophoresis lipoprotein sizes and those obtained by differentialmobility analysis. The scale factor relates 0.86*gel diameter=ionmobility diameter, shifting the mobility sizes of FIG. 5 down by 86% tocorrespond with the lipoprotein classes and subclasses of FIG. 2.

[0128] H. Example of a Typical Sample Run

[0129] Refer now to FIG. 1 depicting the hardware components involved inthe practice of ion mobility analysis of biological particles,specifically lipoproteins. The sample solution comprises 25 milliMolarammonium acetate in an aqueous solution buffered to near neutral pH.

[0130] Between 10 and 50 μL of sample solution is introduced into smallplastic vial, preferably a 1.5 mL microcentrifuge tube 410. Themicrocentrifuge tube 410 has preferably been ultracentrifuged aspreviously described in an ultracentrifuge 420, however, other methodssuch as immunoabsorption or lectin affinity binding can be used toremove the Lp(a) that would otherwise distort the measurement oflipoproteins. The testing microcentrifuge tube 450 is in turn installedin a pressurized chamber 460 on the electrospray generator. About 3 psigof positive pressure is applied to the pressurized chamber 460, forcingthe sample solution 455 to flow through the electrospray capillary 100and out the beveled tip. At the low differential pressure of 3 psig, ittakes several minutes for the sample to fill the capillary and detectsample 455 exiting the beveled tip. An improved alternative method tospeed up filling the capillary is to increase the pressurized chamber460 pressure to about 15 psig, which in turn fills the capillary in onlyabout 30 seconds. When the electrospray capillary is filled, thedifferential pressure is reduced to 3 psig to return the flow rate toabout 50 nL per min.

[0131] Positive high voltage, from a high voltage variable power supply430, is then applied to the sample 455. The high voltage is then scannedfrom ˜5,000 to ˜5,100 volts. The Taylor cone 150 of the capillary 100 isthen examined during a high voltage 430 scan to find the particularvoltage at which the electrospray is stable. A stable electrospray isobtained when the Taylor cone 150 remains fixed in place and pointsdirectly away from the axis of the capillary 100, with a sharp tip and asingle steady stream of material being ejected. Typically, themicroscopic image of the Taylor cone appears as a 45° equilateraltriangle attached to the flat tip on the end of the tapered capillary100.

[0132] About 1.5 L per minute flow of dry gas 210 is used to introducethe droplet stream resulting from the Taylor cone 150 into thedifferential mobility analyzer 200 through connected tubing, afterhaving been first charge reduced with alpha radiation source 480 toeither a neutral or single positive charge state. 15 L per min oflaminar excess gas flow 220 is added to the differential mobilityanalyzer 200. Additional dry gas 230 is introduced to collect themobility selected particles, resulting in a mobility-classified particleflow 240 of 1.5 L per min that exits the differential mobility analyzer200, which in turn transfers the mobility selected particles 240 to thecondensation particle counter 490. By scanning the differential mobilityanalyzer 200 high voltage power supply 205, a data set of particlescounted per second versus mobility may be created.

[0133] Additional confirmation of a stable electrospray is obtained fromknowledge of the electrospray current. The TSI electrospray aerosolgenerator has a current meter for displaying the electrospray currentoutput by the electrospray high voltage power supply 205. Stableelectrosprays exhibit long term stability with currents varying lessthan ±1 nA from a typical total electrospray current of 300 nA duringthe course of an analysis.

[0134] Typically, an essentially pure sample of ammonium acetate buffersolution is run through the instrument to confirm that the capillary isclean. This is called a background spectrum check. Typical backgroundspectra will indicate fewer than 7 particles per mL with particlediameters between 3-4 nm.

[0135] The software supplied with the TSI Model 3980 Gas-phaseElectrophoretic-Mobility Macromolecule/nanoparticle Analysis (GEMMA)unit provides several operator defined conditions. The data recordingsoftware allows several choices for the number of size subdivisionsrecorded for each decade of particle diameter. Typically 64 sizesubdivisions per decade are recorded. The scanning range can also beselected. Typically, a scanning range of 3 nm to 100 nm is scanned fortypical LDL samples; however, this range is extended to a higher limitwhen IDL and chylomicrons are measured, to as high as 1200 nm. Scantimes are typically set to 3 min. The position of a peak in an ionmobility spectrum is determined with the manufacturer's software.Computer mouse movement is used to select the maximum value in a peak byobserving a draggable indicator.

[0136] I. Coronary Heart Disease Risk Analysis

[0137] Differential mobility spectroscopy of lipoproteins canadditionally be used for quick determinations of coronary heart disease(CHD) risk by analyzing the resultant lipoprotein size distributions.

[0138] Plasma was collected from six individuals having lipoproteinpatterns previously characterized by gradient gel electrophoresis.Lipoprotein particles from these individuals were separated from plasmausing ultracentrifugation to isolate components with density less than1.20 g/cm³. The lipoprotein particles were then desalted and analyzedwith ion mobility spectrometry to determine the biological particle sizedistribution. The results for six types of lipoprotein patterns obtainedby electrospray mobility spectroscopy are shown in FIG. 5.

[0139] In FIG. 5, six individual lipoprotein size spectra (510, 520,530, 540, 550, and 560) are presented as the result of differentialmobility analysis with mobilities converted to size as previouslydescribed. In all but the lowest mass value trace 560, the traces areshifted vertically in order to differentiate the graphs, which wouldotherwise overlap. To read the vertically shifted mass value, the traceis shifted vertically to zero mass on its lowest particle diameter. TheMass ordinate is in relatively scaled arbitrary units of grams.

[0140] Pattern A is a designation applied to individuals at relativelylow risk for, CHD. Pattern A lipoproteins are characterized by LDLparticles larger (median size of ˜22.5 nm versus ˜20.8 nm) than thoseobtained from individuals at higher risk for coronary heart disease,i.e., Pattern B individuals. Additionally, Pattern A lipoproteins arecharacterized by a population of HDL particles that generally showsseveral subtypes. These subtypes are revealed by several peaks in theHDL region (of ˜7-13 nm) for Pattern A spectra. The vertical line atapproximately 8.9 nm indicates the principal HDL peak for patients withPattern A, B, and intermediate Pattern AB. The Pattern a spectra areeasily discerned by observing a second HDL peak at approximately 11 nm.With these characteristics in mind, traces 510, 520 and 530 are low riskType A patterns. The peak in the LDL population of particles for PatternB individuals is smaller by several nanometers than LDL particles fromPattern A individuals. The shift from pattern A to pattern B isrepresented approximately by the vertical lines drawn through the LDLpeaks at approximately 20.8 nm (dashed), and 22.5 nm (solid). The HDLparticles from Pattern B individuals, shown in traces 550 and 560,generally show less heterogeneity; here the HDL peaks for Pattern Bpatterns show only one sharp principal peak, as expected. Thus, the ionmobility measurements agree with gradient gel determinations and providean alternative measurement method for assessing CHD.

[0141] Still referring to FIG. 5, trace 540 represents a patient with anintermediate Type AB pattern. This trace 540 shows an LDL peakintermediately disposed between the typical Type A peak at 22.5 nm, andthe typical Type B peak of 20.8 nm. The trace 540 is also distinguishedby only a single HLD peak, typical of the higher risk Type B pattern.

[0142] Ion mobility spectroscopy is quantitative and can be used todirectly measure the total amount of lipoprotein particles in each ofthe lipoprotein classes and/or subclasses. The area under the curves, ina particle mass versus independent variable (such as size, density,mobility, etc.) distribution, is directly representative of thelipoprotein particle mass. The measurement technique relies on countingindividual particles as a function of size (diameter). It is thereforepossible the convert the number of particles at a specific size into amass value using the volume and density of the particles. The density oflipoprotein particles is a well-known function of particle size and isobtainable from the literature. The mass values associated with thefigure are simply scaled to indicate relative values but can beconverted to actual mass of lipoproteins in plasma using dilutionfactors along with flow rates of sample and air passing through the ionmobility spectrometer.

[0143] The spectra in FIG. 5 also show the existence of lipoproteinparticles that fall into the VLDL size class. Their presence isindicated by small bumps in the spectra above particle diameters ofabout 30 nm. The shaded area under the plots in this figure areproportional to particle mass and this area can be used to assess themass of particles in any chosen particle interval such as 30 to 40 nm or35 to 40 nm or any other choice of bin size.

[0144] J. Linearity Versus Density

[0145] A further example showing how ion mobility analysis can beimplemented is shown with the data in FIG. 6. Lipoprotein particles werefractionated with non-equilibrium ultracentrifugation in NaCl/D₂O. Theparticles were removed from the ultracentrifuge tube as liquidfractions, which spanned the range of 1.0 to 1.08 g/cm³. Because theultracentrifugation step was not run until the particles reached anequilibrium flotation position in the ultracentrifuge tube the densityof the lipoprotein particles removed with each fraction is only anestimate, however a reasonable estimate, of the particle's true density,and is displayed as an estimated density. FIG. 6 is a plot oflipoprotein particle diameter as a function of the solution density ofeach fraction reveals the expected inverse linear relationship betweenparticle diameter and density. FIG. 6 has an ordinate of particlediameters in nm as measured by ion mobility analysis, and an abscissa ofestimated particle density in g/cm³ as obtained by non-equilibriumultracentrifugation.

[0146] In FIG. 6, the particle diameter was measured using ion mobilityanalysis. The results indicate, that there is an inverse linearcorrelation between particle diameter and particle density, as expected.Data points not exactly matching the inverse linear correlation arethough to be due to the non-equilibrium floatation method used to obtainthe data samples.

[0147] K. Linearity of Lipoprotein Mass Measurements

[0148] The linearity of the detection method was verified by analyzing aseries of dilutions of a specific set of LDL particles. These LDLparticles were isolated by gel filtration from the other classes oflipoprotein particles. The starting concentration of these LDL particleswas determined by measuring the apolipoprotein B concentration of thesolution. A single molecule of apolipoprotein B is present in each LDLparticle. By using the apolipoprotein B concentration it is possible tocalculate the number of LDL particles in the sample. This specificsample was sequentially diluted, and the number of particles in eachdilution (the IMS peak height, #/cm³ axis) was determined bydifferential mobility spectrometry and plotted versus the calculatedapolipoprotein B concentration (the Particles, #/mL axis) at eachdilution. One option available with the commercial software suppliedwith the instrument manufactured by TSI, Inc., plots the number ofparticles detected per cm³ of air passing through the differentialmobility analyzer. Ion mobility peak-height is plotted vs. the numberconcentration of LDL particles as determined from apolipoprotein Bmeasurements in FIG. 7. This plot, FIG. 7, shows that the measurement islinear over more than three orders of magnitude from 10¹² to 10¹⁴particles per mL. For increased detail, the inset graph of FIG. 7 showsthe range up to 8-10¹² particles per mL. FIG. 7 implies that variationsin concentrations of lipoprotein subclasses will be accurately measuredover a range of 1000:1, or 0.1%.

[0149] L. Linear Independence of Lipoprotein Analyses

[0150] Differential mobility analysis appears to be highly linearlyindependent over the variety of lipoprotein classes and subclasses. Thislinear independence was verified as shown in FIGS. 8-23. In theseFigures, a single human blood sample was fractionated into 16 differentaverage densities. The method used was non-equilibriumultracentrifugation. One byproduct of this type of fractionation is thatthere is always a distribution of densities about an average density.Since we are dealing with biological particles, there are wide ranges ofsizes comprising in the fractions having a particular average densities.An attempt was made to select density fractions representing each of thelipoprotein particle subclasses.

[0151] FIGS. 8-23 show resultant differential mobility analysis scans of16 fractions of human lipoprotein over a range of selected averagedensities spanning the various human lipoprotein classes and subclasses.Lipoprotein particles were prepared as above from human plasma, thensubjected to ultracentrifugation. A density gradient in anultracentrifuge tube was prepared with NaCl dissolved in D₂O. The choiceof gradient composition provided a way to separate lipoprotein particleswith average densities varying between about 1.000 (FIG. 8) and 1.090g/cm³ (FIG. 23) from other material in the plasma. For referencepurposes, gel electrophoresis gradient 8 is used to separate averagedensities over a range from about 1.000 (FIG. 8) to 1.062 g/cm³(bracketed between FIGS. 19 and 20). This density gradient distributeslipoprotein particles along the length of the ultracentrifuge tube.Lipoproteins with higher density such as HDL move to the bottom of theultracentrifuge tube, as do protein molecules. Lower densitylipoproteins such as VLDL rise to the top of the tube during theultracentrifugation process.

[0152] The resulting liquid in the ultracentrifuge tube was distributedinto 16 fractions, which were subsequently dialyzed against 25 mMammonium acetate for 3 hours using a 10,000 molecular weight cut-offdialysis membrane.

[0153] The size distribution of the lipoprotein particles in eachfraction was then determined using ion mobility spectral analysis. Thesesize distributions appear in FIGS. 8-23, and have densities thatcorrespond to lipoprotein classes and subclasses as indicated in VLDL I(FIGS. 8-10) and II (FIG. 11), IDL I (FIG. 12) and II (FIG. 13), and LDLI (FIG. 14), IIa (FIG. 15), IIb (FIG. 16), IIIa (FIG. 17), IVa (FIGS. 18and 19), and HDL IIa (FIGS. 20-23). The fractions of FIGS. 8-23, andtheir subsequent density analyses, are somewhat confusing since theoriginal laboratory sample labels of fractions are respectively 1 a, 1b, 2 a, 2 b, 3-10, 11 a, 11 b, 12 a, and 12 b. Consequently, the samplelabels bear some resemblance to the lipoprotein subclasses. Lipoproteinsubclass IIIb was not shown in this selection of fractions, althoughbased on the complement of FIGS. 8-23, the IIIb distribution should besimilarly peaked within a range of 1.041 and 1.044 g/cm³ as indicated onFIG. 2. Each particle size distribution is plotted as mass vs. particlediameter. In each Figure, the mass value refers to the mass of particlesper m³ at each indicated particle diameter where m³ is referenced to thevolume of lipoprotein-laden gas entering the differential mobilityanalyzer. Although only single positively charged particles are measureddue to the selective electromotive classification used by the DMA, theμg/m³ number is corrected for the fraction of particles that are chargedin any given particle size interval. This rate does not take intoaccount either the rate at which a sample solution is electrosprayed, orthe flow rate of flow gas that sweeps the particles towards the DMA.This mass value can be converted into the mass of lipoprotein particlespresent in the original sample plasma by using the information of thegas flow rate, flow rate of electrosprayed sample, particle transferefficiency and liquid dilution factors.

[0154] FIGS. 9-18, 20, 21, and 23 indicate that for lipoproteindensities ranging from about 1.003-1.090 g/cm³, in this particular humansample, each density sample through ultracentrifugation results in oneprincipal peak in the mass versus size graph.

[0155] For FIGS. 8, 19, and 22 there is some bimodality and broadeningof the peaks. It is not presently understood what these broadened andbimodal lipoprotein distributions represent. It is possible that thisbimodality could be an artifact of the sample preparation methoddiscusses above. That is, the sample preparation technique could bedisruptive enough to break apart some of the agglomerated biologicalparticles, resulting in smaller sized fragments. If this is the case,then a modification of the sample preparation method could then resolvethese samples into relatively clean single peak size spectra as well.

[0156] The bimodality and broadening could also be an artifact resultingfrom the nonequilibrium ultracentrifugation used to generate thesubfractions in this particular experiment. This issue will be resolvedby further experiment.

[0157] Nonequilibrium ultracentrifugation also results in the non-steadystate densities determined in FIGS. 8-23. As such, all densities inthese FIGS. 8-23 are approximate.

[0158] M. Linear Superposition of Lipoprotein Subclasses

[0159] In principal, for lipoproteins represented in these subclasses,an entire plasma sample could be mathematically represented by thelinear superposition of each lipoprotein subclass. Even if, ultimately,the multiple peak phenomenon remains, then traditional numerical methodsof data correlation such as least squares or singular valuedecomposition could be used to determine the amount of each lipoproteinsubclass in an entire sample. Thus, in a single differential mobilityspectral scan, the quantity of each lipoprotein subclass a plasma samplecould be determined. After making this determination, both the quantityand type of each lipoprotein would be known. By statisticallycorrelating the resulting subclass information with population mortalityand risk factors, a more accurate assessment of coronary heart diseaserisk would result. In particular, the known characteristics of a bimodalvs. unimodal HDL distribution, and the peak lipoprotein diameter of theLDL distribution, could be readily transformed into a risk factorranging from 100% Type A pattern (low risk) to 100% Type B pattern (highrisk).

[0160] N. Conclusion

[0161] All publications, patents, and patent applications mentioned inthis specification are herein incorporated by reference to the sameextent as if each individual publication or patent application were eachspecifically and individually indicated to be incorporated by reference.

[0162] The description given here, and best modes of operation of theinvention, are not intended to limit the scope of the invention. Manymodifications, alternative constructions, and equivalents may beemployed without departing from the scope and spirit of the invention.

We claim:
 1. A method for analyzing the size distribution of biologicalparticles, comprising: (a) obtaining a physiological sample comprisingbiological particles; (b) concentrating the biological particles; (c)spraying the biological particles in a charged state; (d) reducing thebiological particles charge state to a uniform charge; (e) deliveringthe charged biological particles to a differential mobility analyzer,whereby the charged biological particles are separated on the basis ofsize; (f) analyzing the biological particles to form a size distributionof the biological particles; and (g) comparing the size distribution ofthe biological particles to a reference size distribution.
 2. The methodof claim 1 wherein said biological particles are lipoprotein particles.3. The method of claim 2 further comprising the step of: analyzing thesize distribution of lipoprotein particles to distinguish between VLDL,IDL, LDL, HDL, and chylomicrons and their subclasses.
 4. The method ofclaim 1 further comprising the step of scanning a selection voltage insaid differential mobility analyzer so that the charged biologicalparticles are subjected to different magnitudes of electrostatic forcesso as to sample biological particles of different sizes.
 5. The methodof claim 1 wherein said analyzing size distribution further comprisesoutputting to a computer readable medium a representation showing numberof particles versus size over the size range of about 1.7 nm to about120 nm.
 6. A method for counting the number biological particles thatpass a detector per second of a given size comprising the steps of: (a)electrospraying a diluted biological sample into a size-selectabledifferential mobility analyzer; (b) forming singly charged biologicalparticle ions; and (c) counting an approximate number of chargedbiological particle ions, dn⁺, in a defined sampling time, dt, in atleast one specific selected size, s, of a selectable differentialmobility analyzer, resulting in a size count rate output of about$\left. \frac{n^{+}}{t} \middle| {}_{S}. \right.$


7. The method of claim 6, further comprising the step of outputting to acomputer readable medium a representation of charged biological particleions counted in the defined sampling time versus size.
 8. The method ofclaim 7 wherein said output is converted to a histogram of chargedlipoprotein particle ions counted in the defined sampling time,$\left. \frac{n^{+}}{t} \right|_{\rho},$

versus density, ρ.
 9. The method of claim 8 wherein said output densityhistogram is assessed for the degree of coronary heart disease risk. 10.The method of claim 6 wherein said diluted biological sample is dilutedto a dilution of one biological particle per electrospray droplet,which, after desolvation, results essentially in an individualbiological particle.
 11. The method of claim 10 wherein said dilutedbiological sample is diluted by ammonium acetate in water.
 12. A methodfor assessing lipid-related health risk comprising the steps of: (a)removing whole blood cells, non-lipoprotein proteins, and Lp(a) from abiological sample, thereby forming a plasma sample; (b) adding to theplasma sample one or more diluents, thereby forming a diluted sample;(c) electrospraying said diluted sample; (d) reducing the electrosprayedsample to a uniform charge; (e) passing said uniformly charged dilutedsample to a volatilizing chamber to evaporate said diluents, formingindividual charged biological particle ions, with a uniform charge; (f)transporting said charged biological particle ions into asize-selectable differential mobility analyzer; (g) counting the numberof charged biological particle ions, dn⁺, in a defined sampling time,dt, at each size selection, s, of the selectable differential mobilityanalyzer, resulting in a size output of$\left. \frac{n^{+}}{t} \right|_{s};{and}$

(h) scanning the size selection over a range of size selectionscomprising the range of 220 to 285 angstroms, resulting in an outputhistogram of charged biological particles counted in the definedsampling time versus size.
 13. The method of claim 12 wherein saidsampling time is held constant, resulting in an output histogram ofcharged biological particle ions, n⁺, versus size.
 14. A method forassessing lipid-related health risk comprising the steps of: (a)removing albumin and whole blood cells from a whole blood sample,thereby forming a plasma sample; (b) diluting said plasma sample withone or more diluents, forming a diluted sample; (c) electrospraying saiddiluted sample, forming electrospray droplets; (d) irradiating saidelectrospray droplets with an alpha radiation source, therebyneutralizing the charge or leaving at most a single positive charge; (e)passing said irradiated electrospray droplets into a volatilizingchamber to evaporate said diluents, forming charged lipoprotein particleions consisting essentially of lipoprotein particles that originated insaid plasma sample; (f) transporting said charged lipoprotein particleions into a size-selectable differential mobility analyzer; (g) countingthe number of said charged particles, dn⁺, in a defined sampling time,dt, at each size selection, s, of said size-selectable differentialmobility analyzer, resulting in a size output of$\left. \frac{n^{+}}{t} \right|_{s};$

(h) converting said size output into a density output of about$\left. \frac{n^{+}}{t} \right|_{\rho};{and}$

(i) scanning the size selection over a range of size selections,resulting in an output histogram of charged particles of at a givendensity counted in the defined sampling time versus density, ρ.
 15. Anapparatus for determining particle size distribution of lipoproteinparticles extracted from a biological sample, comprising: (a) sampledelivery means for delivering a plurality of lipoprotein samples in apredetermined order; (b) an aerosol generator for spraying thelipoprotein sample through a methyl derivatized capillary and convertingthe sample into sprayed particles of uniform charge; (c) a particlemobility analyzer having an exit and a voltage applied to the analyzerwhereby the charged particles exit the differential mobility analyzer asa function of their mobility; (d) a particle counter for countingparticles exiting the particle mobility analyzer; (e) computerized meansfor generating a particle size distribution and for comparing thisdistribution against a known distribution of lipoprotein particles foundin an individual identified as to risk for cardiac heart disease toobtain a risk assessment for the lipoprotein sample; and (f) a computerreadable medium associated with the apparatus containing the riskassessment.
 16. The apparatus of claim 15 wherein said tip is adapted tospray a biological particle size distribution from 2 nm to 120 nm. 17.The apparatus of claim 15 wherein said particle counter of is acondensation counter.
 18. The apparatus of claim 15 wherein saidparticle counter of is a time of flight counter.
 19. The apparatus ofclaim 15 wherein said tip is adapted for spraying aerosol particlesselected from the group consisting of very low density lipoproteins(VLDLs), intermediate density lipoproteins (IDLs), low densitylipoproteins (LDLs), high density lipoproteins (HDLs), and chylomicrons.20. A method for determining particle size distribution in a samplecontaining lipoprotein particles, comprising: (a) preparing the sampleto remove non-lipoprotein contaminants; (b) introducing said sample toan electrospray device, thereby generating a single lipoprotein particleper droplet; (c) forming an aerosol consisting essentially of aplurality of said droplets; (d) introducing said aerosol to a scanningdifferential mobility analyzer, whereby charged droplets are separatedaccording to their mobility in air; (e) counting the particles leavingthe differential mobility analyzer; and (f) acquiring the data fromcounted particles to create a particle size distribution of thelipoprotein particles in the sample, said distribution including therange 19-25 nm.
 21. The method of claim 20 where said sample is humanblood or a component thereof.
 22. The method of claim 20 wherein saiddistribution is further compared to known human lipoprotein profiles inorder to obtain a lipid-related health risk assessment.
 23. The methodof claim 20 wherein said sample is comprised essentially of particlesfrom the group consisting of very low density lipoproteins (VLDLs),intermediate density lipoproteins (IDLs), low density lipoproteins(LDLs), high density lipoproteins (HDLs) and chylomicrons.
 24. Themethod of claim 20 wherein said sample further comprises the step ofcomparing the particle size distribution to pre-determined ranges inorder to assess cardiac risk in a human patient.
 25. A method foranalyzing a size distribution of lipoprotein particles, comprising: (a)obtaining a sample comprising concentrated lipoprotein particles; (b)spraying the biological particles in a uniformly charged state; (c)delivering the charged particles to a differential mobility analyzer,whereby particles are separated on the basis of size; (d) analyzing thesize distribution of the charged particles over a range comprising 72Angstroms to 285 Angstroms, thereby encompassing LDL and HDL particles;and (e) storing data representing the size distribution of thelipoprotein particles in a computer readable medium.
 26. The method ofclaim 25 further comprising the step of analyzing the data stored in thecomputer readable medium to separate the size distribution into at leastone class and its subclasses, selected from the group consisting of:VLDL, IDL, LDL, and HDL.
 27. The method of claim 26 wherein the sizedistribution class is LDL and at least one subclass thereof.
 28. Amethod for determining a cardiovascular disease or condition, or apredisposition to develop said cardiovascular disease or condition of apatient, the method comprising: (a) obtaining a mobility distribution ofcharged lipoprotein particles from blood of the patient; (b) comparingthe mobility distribution of lipoprotein particles from the patient to areference mobility distribution; and (c) determining a cardiovascularcondition of the patient, wherein the mobility distribution of at leastone of the charged lipoprotein particles correlates with said referencemobility distribution to indicate a cardiovascular disease or condition,or a predisposition to develop said cardiovascular disease or condition.29. The method of claim 28, wherein the charged lipoprotein particlescomprise at least one lipoprotein selected from the group consisting ofVLDL, IDL, LDL, HDL, and their subclasses.
 30. The method of claim 28,wherein the charged lipoprotein particles from the patient areclassified on the basis of mobility by a differential mobility analyzer,and the mobility classification is scanned to create a mobilitydistribution of charge reduced lipoprotein particles from the patient.31. The method of claim 28, further comprising: (a) converting themobility distribution of charge reduced lipoprotein particles to atleast one measurement selected from the group consisting of a particlemobility, a particle size, a particle density, a particle mass, a numberof particles in a size interval, and an amount of particle mass in asize interval, prior to determining the cardiovascular condition of thepatient.
 32. The method of claim 28, wherein the charge reducedlipoprotein particles comprise at least one lipoprotein class, includingsubclasses, selected from the group consisting of VLDL, IDL, LDL, andHDL.
 33. A method for determining a level of therapeutic responsivenessto a cardiovascular drug or other therapeutic intervention directed at acardiovascular condition of a patient, comprising: (a) obtaininglipoprotein particles from a patient; (b) obtaining a mobilitydistribution of charge reduced lipoprotein particles; and (c)determining a cardiovascular condition of the patient, wherein themobility distribution of at least one of the charge reduced lipoproteinparticles correlates with the level of therapeutic responsiveness to acardiovascular drug or other therapeutic intervention directed at thecardiovascular condition of the patient.
 34. The method of claim 33,wherein the charged lipoprotein particles comprise at least onelipoprotein selected from the group consisting of VLDL, IDL, LDL, andHDL.
 35. The method of claim 33, wherein the charged lipoproteinparticles from the patient are classified on the basis of mobility by adifferential mobility analyzer, and the mobility classification isscanned to create a mobility distribution of charge reduced lipoproteinparticles from the patient.
 36. A method for determining acardiovascular condition of a patient, the method comprising: (a)obtaining a mobility distribution of charge reduced lipoproteinparticles from the patient; (b) comparing a profile of mobilitydistribution of charge reduced lipoprotein particles with a referenceprofile of mobility distribution of charge reduced lipoproteinparticles, wherein the mobility distribution of at least one of thecharge reduced lipoprotein particles correlates with a cardiovasculardisease or condition, or a predisposition to develop said cardiovasculardisease or condition; and (c) determining the cardiovascular conditionof the patient.
 37. The method of claim 36, further comprisingconverting the mobility distribution of charge reduced lipoproteinparticles to a mass distribution profile of lipoprotein particles andcomparing with a reference profile of mass distribution of lipoproteinparticles, wherein the reference profile of mass distribution correlateswith a cardiovascular disease or condition, or a predisposition todevelop said cardiovascular disease or condition.
 38. The method ofclaim 36, wherein at least one of the steps of (b) the comparison of theprofiles of mobility distribution of charge reduced lipoproteinparticles and (c) the determination of the cardiovascular condition ofthe patient, is carried out by a deterministic algorithm.
 39. The methodof claim 36, wherein the charge reduced lipoprotein particles compriseat least one lipoprotein selected from the group consisting of VLDL,IDL, LDL, HDL chylomicrons.
 40. A method for determining acardiovascular condition of a patient, the method comprising: (a)obtaining a mobility distribution of charge reduced lipoproteinparticles from the patient; (b) converting the mobility distribution ofcharge reduced lipoprotein particles to at least one measurementselected from the group consisting of a particle mobility, a particlesize, a particle density, a particle mass, a number of particles in asize interval, and an amount of particle mass in a size interval (c)comparing a profile of the measurement(s) from step (b) with a standardprofile of the measurement(s), wherein the measurement(s) correlateswith a cardiovascular disease or condition, or a predisposition todevelop said cardiovascular disease or condition; and (d) determiningthe cardiovascular condition of the patient.
 41. The method of claim 40,wherein the charge reduced lipoprotein particles comprise at least onelipoprotein selected from the group consisting of VLDL, IDL, LDL, HDL,and chylomicrons.
 42. A preparation of biological particles comprisingcharge reduced biological particles suspended in air, wherein thebiological particles are isolated from a physiological sample.
 43. Thepreparation of claim 42, wherein the biological particles are selectedfrom the group consisting of: a lipoprotein particle, a viral particle,and an immune complex.
 44. The preparation of claim 42, wherein thebiological particles are lipoprotein particles prepared fromlipoproteins isolated from a single patient.
 45. The preparation ofclaim 44, wherein the charge reduced lipoprotein particles comprise atleast one lipoprotein selected from the group consisting of VLDL, IDL,LDL, HDL and chylomicrons.
 46. The preparation of claim 42, wherein thecharge reduced lipoprotein particles have a charge state that ispredominantly neutral or single positive charge.
 47. The preparation ofclaim 42, wherein the lipoprotein particles are prepared fromlipoproteins isolated from a blood, a serum or a plasma of the patient.48. A biological particle droplet suspended in air, comprising (i) abiological particle, (ii) an excess charge, and (iii) a volatile bufferhaving a pH wherein the biological particle is structurally stable. 49.The droplet of claim 48, wherein the biological particles are selectedfrom the group consisting of: a lipoprotein particle, a viral particle,and an immune complex.
 50. The droplet of claim 48, wherein thebiological particle is a lipoprotein particle prepared from lipoproteinsisolated from a single patient.
 51. The droplet of claim 50, wherein thecharged lipoprotein particle is selected from the group consisting ofVLDL, IDL, LDL, HDL, and their subclasses, and chylomicrons.
 52. Thedroplet of claim 48, wherein the charge reduced lipoprotein particleshave a charge state that is predominantly neutral or single positivecharge.
 53. The droplet of claim 48, wherein the lipoprotein particle isprepared from lipoproteins isolated from a blood, a serum, or a plasmaof the patient.
 54. The droplet of claim 48, wherein the buffer is avolatile aqueous buffer at near neutral pH.
 55. A method for determininga size distribution of lipoprotein particles in a patient sample, themethod comprising: (a) spraying a mixture of a plurality of thelipoprotein particles and a diluent, the mixture sprayed in a chargedstate; (b) drying the diluent from the mixture, whereby the driedmixture contains essentially only the biological particles; (c) reducingthe charge state of the biological particles to a charge state that ispredominantly a neutral or a single positive charge; (d) delivering thecharge reduced biological particles to a differential mobility analyzer(DMA), whereby the charge reduced biological particles are classified onthe basis of mobility; (e) detecting the mobility classified chargereduced biological particles; and (f) scanning the mobilityclassification of the DMA to create a size distribution of lipoproteinparticles in the patient.
 56. The method of claim 55, wherein the sizedistribution comprises at least one measurement selected from the groupconsisting of a particle mobility, a particle size, a particle density,a particle mass, a number of particles in a size interval, and an amountof particle mass in a size interval.
 57. The method of claim 56, furthercomprising: (a) correlating the size distribution of lipoproteinparticles from the patient with a cardiovascular disease or condition,or a predisposition to develop said cardiovascular disease or condition.